Polymerase Chain Reaction

(PCR)
Outline
1. DNA
2. PCR
– Targets
– Denaturing
– Primers
– Annealing
– Cycles
– Requirements
Outline
3. Applications of PCR
– Neisseria gonorrhoeae
– Chlamydia
– HIV-1
– Factor V Leiden
– Forensic testing
4. Extraction of DNA for Factor V
Outline
5. DNA Detection for Factor V
6. PCR Results for Factor V
7. Conclusion
DNA
DNA is a nucleic acid that is composed of two
complementary nucleotide building block
chains.

The nucleotides are made up of a phosphate

group, a five carbon sugar, and a nitrogen
base.
DNA
• DNA Sugar
– Deoxyribonucleic acid

• RNA Sugar
– Ribonucleic acid
DNA
DNA has four nitrogen bases.

• Two are purines ( 2 ringed base )

– Adenine ( A ), Guanine ( G )

• Two are pyrimidines ( 1 ringed base )

– Cytosine ( C ), Thymine ( T )
DNA
These four bases are linked in a repeated
pattern by hydrogen bonding between the
nitrogen bases.

The linking of the two complementary

strands is called hybridization.
DNA
A purine always links with a pyrimidine base to maintain
the structure of DNA.

Adenine ( A ) binds to Thymine ( T ), with two hydrogen

bonds between them.

Guanine ( G ) binds to Cytosine ( C ), with three hydrogen

bonds between them.
DNA
Example of bonding pattern.
• Primary strand

CCGAATGGGATGC
GGCTTACCCTACG

• Complementary strand
DNA Molecule
Adenine

Thymine

Guanine

Cytosine
PCR
PCR is a technique that takes a specific
sequence of DNA of small amounts and
amplifies it to be used for further testing.
PCR Targets
The targets in PCR are the sequences of DNA
on each end of the region of interest, which
can be a complete gene or small sequence.
PCR Targets
The number of bases in the targets can vary.

TTAAGGCTCGA . . . . AATTGGTTAA

The . . . . Represents the middle DNA sequence,

and does not have to be known to replicate it.
PCR Denaturing
Denaturing is the first step in PCR, in which

the DNA strands are separated by heating to

95°C.
PCR Primers
Primers range from 15 to 30 nucleotides, are
single-stranded, and are used for the
complementary building blocks of the target
sequence.
PCR Primers
A primer for each target sequence on the end
of your DNA is needed. This allows both
strands to be copied simultaneously in both
directions.
PCR Primers

TTAACGGCCTTAA . . . TTTAAACCGGTT
AATTGCCGGAATT . . . . . . . . . .>
and
<. . . . . . . . . . AAATTTGGCCAA
TTAACGGCCTTAA . . . TTTAAACCGGTT
PCR Primers
The primers are added in excess so they will
bind to the target DNA instead of the two
strands binding back to each other.
PCR Annealing
Annealing is the process of allowing two
sequences of DNA to form hydrogen bonds.
The annealing of the target sequences and
primers is done by cooling the DNA to 55°C.
PCR Taq DNA Polymerase
Taq stands for Thermus aquaticus, which is a
microbe found in 176°F hot springs in Yellow
Stone National Forest.
PCR Taq DNA Polymerase
Taq produces an enzyme called DNA
polymerase, that amplifies the DNA from the
primers by the polymerase chain reaction, in
the presence of Mg.
PCR Cycles
PCR Cycles
PCR Cycles
PCR Cycles
PCR Cycles
PCR Cycles Review
• Denaturalization: 94°- 95°C

• Primer Annealing: 55°- 65°C

• Extension of DNA: 72°

• Number of Cycles: 25-40

PCR Requirements
• Magnesium chloride: .5-2.5mM
• Buffer: pH 8.3-8.8

• dNTPs: 20-200µM
• Primers: 0.1-0.5µM
• DNA Polymerase: 1-2.5 units

• Target DNA: ≤ 1 µg
Applications of PCR
• Neisseria gonorrhea
• Chlamydia trachomatis
• HIV-1
• Factor V Leiden
• Forensic testing and many others
Applications of PCR
Neisseria gonorrhea and Chlamydia
trachomatis are two of the most common
sexually transmitted diseases. The
infections are asymptomatic and can lead
to pelvic inflammatory disease, salpingitis
in women, epididymitis in men, infertility,
and ectopic pregnancy.
Applications of PCR
Specimens include endocervical
swabs,urethral swabs, and urine samples.

The swabs are placed in a vial with transport

buffer containing ≥ 50mM MgCL2 and
sodium azide as a preservative.
Applications of PCR
The swab specimens can be stored 2-30°C for
4 days or frozen at -20°C.
The urine samples are refrigerated at 2-8°C
or stored at -20°C.
A target sequence is chosen for both,
amplified with polymerase, and then
evaluated with an enzyme immunoassay.
Applications of PCR
HIV-1 and Factor V Leiden also have a specific
target sequence amplified, and then
quantitated by using a microwell probe,
horse-radish peroxidase enzyme, and
chromogen substrate.
Applications of PCR
The HIV-1 test is used as a monitor of the
severity of the virus. The HIV-1 causes a
depletion of CD4+ T lymphocytes, causing
immunodeficiency, multiple opportunistic
infections, malignancies, and death.
Applications of PCR
The HIV-1 specimen is plasma collected in
EDTA that must be separated from the cells
within 6 hours.

Heparin cannot be used as an anticoagulant

because it inhibits PCR.
Applications of PCR
A 142 base target sequence in the HIV-1 gag
gene is converted from RNA to
complementary DNA, and to double
stranded DNA using Thermus thermophilus
DNA polymerase in the presence of
manganese and buffers, which performs
the reverse transcription and the
amplification steps simultaneously.
Applications of PCR
The standard specimen procedure can
quantitate HIV-1 RNA in a range of 400-
75,000 copies/mL.
Applications of PCR
Factor V Leiden is the Factor V in the
coagulation cascade.
Factor V is a genetic point mutation that
causes increased risk of life-threatening
blood clots.
The mutation causes the Factor V molecule to
be unresponsive to the natural anti-
coagulant protein C.
Applications of PCR
Factor V Leiden shifts the patient’s
hemostatic balance to thrombosis.

Factor V mutation gives an increase risk of

venous thrombosis in a homozygous
person, during pregnancy, surgery, or while
using oral contraceptives.
Applications of PCR
Thrombosis - is the development of a blood clot that
occurs in 20-40% of patients with venous
thrombosis.
Thrombophilia - a tendency towards clotting that
occurs in 40-65% of adults with unexplained
thrombophilia.
Protein C - a naturally occurring anti-
coagulant that occurs in 95-100% of people with
activated protein C resistance.
Application of PCR
Treatment for patients with Factor V Leiden
mutations are to give lifelong coumadin.

Women with the mutation should not take

oral contraceptives, and they have
increased risk of thrombosis during
pregnancy.
Applications of PCR
PCR can also be used in forensic testing.

The DNA sequences used are of short

repeating patterns called VNTR (variable
number of tandem repeat), which can
range from 4 to 40 nucleotides in different
individuals.
Applications of PCR
One set of VNTR locus are inherited from the
mother and one set from the father.
The genes are amplified using PCR, and then
run through electrophoresis.
The position of the two bands on the
electrophoresis gel depends on the exact
number of repeats at the locus.
Applications of PCR
Applications of PCR
Three VNTR loci from suspects, along with the
DNA from the scene are run through PCR
amplification, and then through
electrophoresis.
This gives six bands, which can have common
bands for some individuals, but the overall
pattern is distinctive for each person.
Applications of PCR
Extraction of DNA for Factor V
The anticoagulant tube with the patient’s
blood sample should be centrifuged to
separate it into the layers of plasma, Buffy
coat, and the RBCs.

The buffy coat is used for the extraction

because it contains WBCs, which are
nucleated and possess the DNA.
Extraction of DNA for Factor V
Extract and discard plasma, taking care not to remove
the buffy coat.
Extraction of DNA for Factor V
Carefully extract 200µl of buffy coat from each sample
and place in designated tube.
Extraction of DNA for Factor V
Add 25µl of
protease
to each tube.

Add 200µl of
lysis buffer
to each tube.
Extraction of DNA for Factor V
Vortex each tube
for 15 sec. to
ensure proper
mixing.
Extraction of DNA for Factor V

Incubate each tube

for 10 min. at 56°C.
Extraction of DNA for Factor V
Centrifuge each to Add 210µl of ethanol,
remove any mixture vortex and then
that may be on the lid. centrifuge again.
Extraction of DNA for Factor V
Add sample mixture to column tube and centrifuge
for 1 min.
Extraction of DNA for Factor V
Column Tube
This column section fits into
another tube, which catches
the eluted substances.
This is the
column.

This is the eluted

sample that is
discarded.
Extraction of DNA for Factor V
The column portion is inserted into a new tube and
washed twice, each time 500µl of buffer is used to elute
substances
adsorbed to
the column
that are not
DNA.
Extraction of DNA for Factor V
The column portion is then centrifuged for 1
minute to remove excess washing buffer.

Next, 100µ L of eluting buffer is added.

• This is incubated for 5 minutes at 25°C, and
then centrifuged.
• The elute is kept this time because it contains
the DNA.
Extraction of DNA for Factor V
During the 5 minute incubation, the master mix should be prepared.
Master Mix
•10x Buffer - 10 µl
•MgCl - 6 µl
•dNTP mix - 0.8 µl
of each nucleotide
•F5F primer - 2 µl
•F5R primer - 2 µl
•Taq polymerase - 0.5 µl
•Sterile HO - 73.7 µl
 Extraction of DNA for Factor V
Place 5µl of patient sample
and 95µl of master mix in
vials and place these vials
in a PCR panel, which
will then be placed in
the thermocycler for the
DNA amplification cycles.
DNA Detection for Factor V
To prove that the DNA was amplified a DNA
enzyme immunoassay (DEIA) is performed.
The test is done by denaturing the amplified
DNA and adding it to probe-coated microtiter
wells.
If the amplified DNA sequences are
complementary to the probes, double
stranded hybrids will form.
DNA Detection for Factor V
A mouse monoclonal antibody is added that will only
bind to double-stranded DNA hybrids.
Positive and negative wells are detected
colorimetric by adding an enzyme (conjugated
protein A with horseradish peroxidase), substrate,
and chromogen.
The is incubated at room temp. away from light for
30 mins. to develop the color.
 DNA Detection for Factor V
Upon finishing the
incubation, the
panel looks like
this.
 DNA Detection for Factor V
The color is then
stopped, by the
addition of
200µl of an
acidic stop
solution.
DNA Detection for Factor V
The plate is then placed in the automated reader,
where each well is read spectrophotometrically.
DNA Detection for Factor V
Each well is read at 450nm and then at 630nm.
The difference between the two absorbance:
A at 450nm
- A at 630nm
Final A value

A positive hybridization result is indicated by an

absorbance value greater that the mean
negative control plus 0.150 absorbance units.
DNA Detection for Factor V
The machine
then gives
you a read out,
from which
you calculate
the patient
results.
DNA Detection for Factor V
Obtain a ratio
from the values:
0.982
0.041
which will give
you
23.95

Homozygous
Normal = >5.0
Conclusion
PCR is not only vital in the clinical laboratory by
amplifying small amounts of DNA for STD
detection, but it is also important for genetic
predisposing for defects such as Factor V Leiden.
The PCR technology can also be employed in law
enforcement, genetic testing of animal stocks
and vegetable hybrids, and drug screening along
with many more areas.
References
• Assay: Abbott Laboratories
Neisseria gonorrhoeae
List # 8A48-81
• Assay: Roche Diagnostics
Amplicor HIV-1 Monitor Test
List# 83088
• Assay: GEN-ETI-K (DiaSorin)
DEIA-Factor V Leiden
Catalog# PS5096
References
• Alberts, Brown,Johnson, Lewis, Raff, Roberts, Walter. Use
of PCR in Forensic Science. 1998. Online. Internet. 18 Jan.
2001. Available http://www.accessexcellence.org/AB/GG/
forensci_PCR.htm.l
• Brown, John C. What The Heck Is PCR? 1995. Online.
Internet. 18 Jan. 2001. Available
http://falcon.cc.ukans.edu/~jbrown/pcr.html
• Photographs: Courtesy of UMC clinical lab and Tom
Wiggers.
References
• Ronald H. Holton, Ph.D.:
– Molecular Diagnostics in the Clinical
Laboratory
– Molecular Biology in the Clinical Laboratory
– Molecular Pathology: Basic Methodologies
and Clinical Applications
– Expanding applications of PCR, by Peter
Gwynne and Guy Page